PROJECT SUMMARY Osteoclasts acidify the resorptive microenvironment and digest the organic matrix of bone via secretion of lysosomes through the ruffled border circumscribed by a tight sealing zone (actin ring). RANKL is a seminal cytokine for osteoclast differentiation. In addition, RANKL directly stimulates the bone-resorbing activity of mature osteoclasts in concert with immunoreceptor and integrin signaling pathways. While the molecular mechanisms regulating osteoclast cytoskeleton and actin-ring formation are well understood, how osteoclast activating signals stimulate lysosome secretion during bone resorption and how lysosomes are linked to and transported along the microtubules are completely unknown. The work from us and others has implicated PLEKHM1 as an indispensable regulator of intracellular lysosome trafficking and secretion in osteoclasts. By phospho-proteomic, we found that PLEKHM1 S491 and Y991, putatively phosphorylated by RANKL or immunoreceptor signaling and c-Src, respectively, are critical for osteoclast bone resorption. Moreover, PLEKHM1 interacts with TAK1 and RAP1B - two key molecules of the RANKL and integrin signaling pathways. Additionally, we found that PLEKHM1 forms a ternary complex with DEF8 and RAB7 at lysosomes. PLEKHM1 also interacts with FAM98A, and NDEL1 in osteoclasts. FAM98A has a conservative tubulin-binding domain that mediates the docking of transported cargoes or organelles onto the microtubules; and that NDEL1 interacts with cytoplasmic dynein and/or kinesins to promote organelle transport along the microtubules. Based on these lines of premise, we hypothesize that osteoclast activating signals derived from RANKL, M-CSF, immunoreceptors, and/or integrins stimulate lysosome secretion, independently of their role in actin ring formation, by phosphorylating and activating PLEKHM1. Active PLEKHM1 interacts with microtubule-associate proteins FAM98A and NDEL1, thereby, links lysosomes to microtubules and facilitates lysosome transportation to the ruffled border. FAM98A and NDEL1 play an important role in bone resorption and bone remodeling in vivo. To test these interrelated hypotheses, we will (a) Define the signaling cascades that phosphorylate and activate PLEKHM1 and how they stimulate lysosome secretion in osteoclasts (Aim1); (b) Determine the role of PLEKHM1-FAM98A and PLEKHM1-NDEL1 interactions in lysosome secretion in osteoclasts and bone homeostasis in mice. (Aim2). Successful completion of the proposed work should advance our understanding of critical aspects of osteoclast biology, namely the specific role of RANKL, M-CSF, immunoreceptors, and/or integrin signaling in lysosome secretion and the molecular basis of microtubule-dependent lysosome transportation in osteoclasts. In view of the evidence that PLEKHM1 is indispensable for lysosome trafficking in osteoclasts, but not in other cell types, elucidation of the molecular mechanisms by which PLEKHM1 and its interacting proteins regulate lysosome secretion may identify osteoclast-specific candidate therapeutic targets for metabolic bone diseases.